PROJECT SUMMARY A sterilizing cure for HIV infection has remained elusive and persists as one of the greatest challenges in the field. The rapid mutation rate of the virus and its subsequent escape from recognition by the adaptive immune system has hindered not only prophylactic vaccination strategies, but also eradication of the virus from the bodies of infected individuals, thus necessitating lifelong antiretroviral therapy. This viral ?escape? from immunorecognition continues to pose a tremendous challenge for research efforts aimed at inducing antiviral immunity via cytotoxic T lymphocytes (CTLs) and antibodies (Abs), both prophylactically and therapeutically. In this study, we will develop and evaluate the in vitro efficacy of a novel immunotherapeutic cure strategy using the SIVmac239 model of HIV infection. We seek to induce constitutive CTL-mediated killing of SIVmac239- infected cells via a bifunctional fusion protein containing monoclonal Ab (mAb) and peptide-loaded MHC I (pMHCI) domains. The mAb domain will be responsible for fusion protein localization to the infected cell surface, while the pMHCI domain will recruit and activate CTLs to kill the infected cell. In Aim 1 of the proposed study, we will evaluate the use of SIVmac239 envelope (Env)-binding mAbs for targeting and marking SIVmac239- infected cells. Fifteen SIVmac239 Env-binding mAbs will be screened for their ability to bind infected cells and for their susceptibility to viral escape. These mAbs include the known infected cell-binders eCD4-Ig and 5L7 IgG, along with 13 novel Env-binding mAbs isolated from infected animals by the Watkins laboratory. In Aim 2, we will produce fusion proteins composed of a SIVmac239 Env-binding mAb and a MHC I molecule loaded with an immunodominant CTL epitope, then test the ability of these fusion proteins to induce CTL-mediated killing of infected cells in vitro. We will begin our fusion protein studies with eCD4-Ig and 5L7 IgG, both of which bind infected cells and do not appear to select for escape mutants based on previous studies. We aim to recruit high- frequency CTL populations ( > 10% of entire host CTL repertoire), and will therefore include the immunodominant epitopes SIVmac239 Tat SL8 and rhesus cytomegalovirus (RhCMV) IE1 VY9 in our fusion proteins. By recruiting abundant and ubiquitous CTL populations to kill infected cells, we hypothesize that these fusion proteins could provide robust and long-term suppression of viral replication, if not sterilization. Importantly, this strategy would allow exogenous delivery of MHC I molecules loaded with invariant peptide antigens that are independent of viral genotype. Thus, these fusion proteins would induce constitutive killing of SIVmac239-infected cells, regardless of whether the virus harbors CTL escape mutations. This study will provide insight into mAb-mediated targeting of HIV/SIV-infected cells, the potency and specificity of antiretroviral CTL responses, and the therapeutic challenge of CTL escape, all of which will facilitate the future development of antiretroviral immunotherapies. Most importantly, the results of this study could provide proof-of-concept for pMHCI-mAb fusion proteins as a novel strategy for treating and potentially sterilizing HIV/SIV infection.